U.S. patent application number 12/278100 was filed with the patent office on 2009-12-10 for method of treatment for muscular dystrophy.
This patent application is currently assigned to Giulio Cossu. Invention is credited to Silvia Brunelli, Emilio Clementi, Giulio Cossu.
Application Number | 20090304815 12/278100 |
Document ID | / |
Family ID | 37998295 |
Filed Date | 2009-12-10 |
United States Patent
Application |
20090304815 |
Kind Code |
A1 |
Cossu; Giulio ; et
al. |
December 10, 2009 |
METHOD OF TREATMENT FOR MUSCULAR DYSTROPHY
Abstract
The invention relates to a method of treatment for muscular
dystrophies, including Duchenne, Becker, limb-girdle,
facioscapulohumeral, congenital muscular dystrophies and the like
using a combination of nitric oxide-releasing and anti-inflammatory
compounds.
Inventors: |
Cossu; Giulio; (Milano,
IT) ; Clementi; Emilio; (Milano, IT) ;
Brunelli; Silvia; (Milano, IT) |
Correspondence
Address: |
Kenneth K Sharples;Law Office of Kenneth K Sharples
Sena Plaza Building Suite 54 Second Floor, 125 East Palace Avenue
Santa Fe
NM
87501
US
|
Assignee: |
Cossu; Giulio
Milano
IT
Clementi; Emilio
Milano
IT
Brunelli; Silvia
Milano
IT
|
Family ID: |
37998295 |
Appl. No.: |
12/278100 |
Filed: |
February 1, 2007 |
PCT Filed: |
February 1, 2007 |
PCT NO: |
PCT/EP07/00858 |
371 Date: |
April 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60765135 |
Feb 3, 2006 |
|
|
|
Current U.S.
Class: |
424/646 ;
514/165; 514/236.2; 514/263.34; 514/355; 514/470; 514/509; 514/561;
514/562; 514/706; 514/742 |
Current CPC
Class: |
A61K 31/34 20130101;
A61K 31/34 20130101; A61K 31/192 20130101; A61P 21/00 20180101;
A61K 31/60 20130101; A61K 2300/00 20130101; A61K 31/60 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/192 20130101;
A61K 45/06 20130101 |
Class at
Publication: |
424/646 ;
514/509; 514/706; 514/561; 514/742; 514/470; 514/562; 514/236.2;
514/355; 514/165; 514/263.34 |
International
Class: |
A61K 33/26 20060101
A61K033/26; A61K 31/21 20060101 A61K031/21; A61K 31/095 20060101
A61K031/095; A61K 31/195 20060101 A61K031/195; A61K 31/04 20060101
A61K031/04; A61K 31/34 20060101 A61K031/34; A61K 31/5377 20060101
A61K031/5377; A61K 31/44 20060101 A61K031/44; A61K 31/60 20060101
A61K031/60; A61K 31/522 20060101 A61K031/522; A61P 21/00 20060101
A61P021/00 |
Claims
1- Use of a combination of at least one nitric oxide releasing
compound and at least one anti-inflammatory compound for the
preparation of a medicament or a diet supplement for the treatment
or prevention of muscular dystrophy.
2- The use according to claim 1 wherein the nitric oxide releasing
compound is selected from the group of: organic nitrates,
spontaneous nitric oxide donors, nitrosothiols or nitric oxide
synthesis precursors.
3- The use according to claim 2 wherein the nitric oxide synthesis
precursor is L-arginine.
4- The use according to claim 1 wherein the nitric oxide releasing
compound is selected from the group of: sodium nitroprusside,
nitroglycerin, isosorbide dinitrate, isosorbide mononitrate, amyl
nitrite, gallium nitrate, S-nitroso acetylpenicillamine,
molsidomine, nicorandil, or derivatives thereof.
5- The use according to any previous claims wherein the
anti-inflammatory compound is a non-steroidal anti-inflammatory
compound.
6- The use according to claim 5 wherein the non-steroidal
anti-inflammatory compound is selected from the group of:
salicylates and derivatives thereof, para-aminophenol derivatives,
acetic acid derivatives, fenamates, propionic acid derivatives,
enolic acids and derivatives thereof, pyrazolon derivatives,
sulfonanilide derivatives, cyclooxygenase-2 selective
inhibitors.
7- The use according to claim 5 wherein the non-steroidal
anti-inflammatory compound is selected from the group of: aspirin,
salsalate, diflunisal, ibuprofen, acetaminophen, flurbiprofen,
ketoprofen, piroxicam, meloxicam, nabumetone, naproxen, fenoprofen,
diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac,
oxaprozin, flufenamic acid, meclofenamate, mefenamic acid,
celecoxib, valdecoxib, nimesulide, as R or S enantiomer or as
racemate.
8- The use according to claims 1 to 4 wherein the anti-inflammatory
compound is a methylxanthine anti-inflammatory compound.
9- The use according to claim 1 wherein the nitric oxide releasing
compound is isosorbide mononitrate and the anti-inflammatory
compound is aspirin.
10- The use according to claim 1 wherein the nitric oxide releasing
compound is isosorbide dinitrate and the anti-inflammatory compound
is ibuprofen, as R or S enantiomer or as racemate.
11- The use according to any previous claims wherein the muscular
dystrophy is selected from the group of: Duchenne muscular
dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular
dystrophy, myotonic muscular dystrophy, limb-girdle muscular
dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss
muscular dystrophy, distal muscular dystrophy or congenital
muscular dystrophy.
12- A pharmaceutical composition comprising an effective amount of
at least one nitric oxide releasing compound and at least one
anti-inflammatory compound according to claims 1 to 10 and diluents
and/or emollients and/or adjuvants and/or eccipients.
13- A diet supplement comprising an effective amount of at least
one nitric oxide releasing compound and at least one
anti-inflammatory compound according to claims 1 to 10 and diluents
and/or adjuvants and/or eccipients.
14- A method of treating or preventing muscular dystrophy
comprising administering the composition according to claim 12 or
the diet supplement according to claim 13.
Description
[0001] The invention relates to a method of treatment for muscular
dystrophies, including Duchenne, Becker, limb-girdle,
facioscapulohumeral, congenital muscular dystrophies and the like
using a combination of anti-inflammatory and nitric oxide-releasing
compounds.
BACKGROUND OF THE INVENTION
[0002] The muscular dystrophies (MD) are a group of more than 30
genetic diseases characterized by progressive weakness and
degeneration of the skeletal muscles that control movement. Some
forms of MD are seen in infancy or childhood, while others may not
appear until middle age or later. The disorders differ in terms of
the distribution and extent of muscle weakness (some forms of MD
also affect cardiac muscle), age of onset, rate of progression, and
pattern of inheritance. Duchenne MD is the most common form of MD
and primarily affects boys. It is caused by the absence of
dystrophin, a protein involved in maintaining the integrity of
muscle. Onset is between 3 and 5 years and the disorder progresses
rapidly. Most boys are unable to walk by age 12, and later need a
respirator to breathe. Girls in these families have a 50 percent
chance of inheriting and passing the defective gene to their
children. Boys with Becker MD (very similar to but less severe than
Duchenne MD) have faulty or not enough dystrophin.
Facioscapulohumeral MD usually begins in the teenage years. It
causes progressive weakness in muscles of the face, arms, legs, and
around the shoulders and chest. It progresses slowly and can vary
in symptoms from mild to disabling. Myotonic MD is the disorder's
most common adult form and is typified by prolonged muscle spasms,
cataracts, cardiac abnormalities, and endocrine disturbances.
Individuals with myotonic MD have long, thin faces, drooping
eyelids, and a swan-like neck. Muscular dystrophies are caused by
progressive degeneration of skeletal muscle fibres. Lack of one of
several proteins located either at the plasma membrane [1] or, less
frequently, within internal membranes [2] increases the probability
of damage during contraction, and eventually leads to fibre
degeneration, accompanied by severe local inflammation with
infiltration of immune-competent cells [3,4]. In the most severe
forms, such as Duchenne Muscular Dystrophy, regeneration is
exhausted and skeletal muscle is progressively replaced by fat and
fibrous tissue. This condition leads the patient to progressive
weakness and eventually death by respiratory and/or cardiac
failure.
[0003] The current therapeutic approaches to muscular dystrophies
involve steroids, such as prednisolone and deflazacort, and
dantrolene, administered in various protocols. These treatments
result in modest beneficial effects and are accompanied by severe
side-effects including osteoporosis, hypertension, Cushing
syndrome, weight gain, cataracts, short stature, gastrointestinal
symptoms, behavioural changes in the case of steroids, and liver
damage with dantrolene [5-7]. With steroids, moreover there is no
consensus regarding their use as standard treatment. Alternative
therapeutic approaches to muscular dystrophies have been proposed,
and various compounds and drugs tested. None of them has yielded
favourable outcomes in clinical trials and entered the clinical
practice [5].
[0004] Thus there is a need to identify therapeutic agents which
slow the muscle fibre damage and delay the onset of disability in
patients with muscular dystrophies more efficiently than the
current therapies and causing a lesser degree of side effects. The
beneficial effect of corticosteroids is believed to reside in the
blocking of immune cell activation and infiltration which are
precipitated by muscle fibre damage resulting from the disease
[3,4]. Nitric oxide (NO) generated by a muscular NO synthase
structurally and functionally linked to the dystrophin complex at
the sarcolemma participates to physiological development and
function of skeletal muscle. In particular it regulates
excitation-contraction coupling, in such a way that it prevents the
muscle from being damaged during its contractile activity. In
addition, NO couples muscle function to energy availability by
promoting vasodilation and thus supply of oxygen during exercise,
by increasing glucose uptake in the myofibres, and by regulating
the activity of enzymes relevant to cell energy metabolism
[8-9].
[0005] NSAIDs are known to reduce inflammation through inhibition
of cyclooxygenases 1 and 2 [10]. The authors found that the
combination of nitric oxide-releasing compounds and NSAIDs is
effective in retarding the development of, or reversing muscular
dystrophies. Moreover these compounds, for which a novel use is
proposed, are known to cause less side effects than current
therapies for muscular dystrophy. They are in general well
tolerated by the patients and are suitable for long-term
therapies.
SUMMARY OF THE INVENTION
[0006] The present invention provides an effective and safe
treatment for muscular dystrophies with less side-effects by using
a combination of anti-inflammatory drugs and NO-releasing
compounds.
[0007] Therefore it is an object of the present invention the use
of a combination of at least one nitric oxide releasing compound
and at least one anti-inflammatory compound for the preparation of
a medicament or a diet supplement for the treatment or prevention
of muscular dystrophy. Preferably the nitric oxide releasing
compound is selected from the group of: organic nitrates,
spontaneous nitric oxide donors, nitrosothiols or nitric oxide
synthesis precursors. More preferably the nitric oxide synthesis
precursor is L-arginine. In a preferred embodiment the nitric oxide
releasing compound is selected from the group of: sodium
nitroprusside, nitroglycerin, isosorbide dinitrate, isosorbide
mononitrate, amyl nitrite, gallium nitrate, S-nitroso
acetylpenicillamine, molsidomine, nicorandil, or derivatives
thereof.
[0008] In another particular embodiment the anti-inflammatory
compound is a non-steroidal anti-inflammatory compound. Preferably
the non-steroidal anti-inflammatory compound is selected from the
group of: salicylates and derivatives thereof, para-aminophenol
derivatives, acetic acid derivatives, fenamates, propionic acid
derivatives, enolic acids and derivatives thereof, pyrazolon
derivatives, sulfonanilide derivatives, cyclooxygenase-2 selective
inhibitors. More preferably the non-steroidal anti-inflammatory
compound is selected from the group of: aspirin, salsalate,
diflunisal, ibuprofen, acetaminophen, flurbiprofen, ketoprofen,
piroxicam, meloxicam, nabumetone, naproxen, fenoprofen, diclofenac,
indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin,
flufenamic acid, meclofenamate, mefenamic acid, celecoxib,
valdecoxib, nimesulide, as R or S enantiomer or as racemate. These
compounds can be used either as racemate or R or S enantiomer, for
instance (R,S)-Flurbiprofen, (R)-Flurbiprofen, (S)-Flurbiprofen;
(R,S)-Ibuprofen, (R)-Ibuprofen, (S)-ibuprofen; (R,S)-Fenoprofen,
(R)-Fenoprofen, (S)-fenoprofen; (R,S)-naproxen, (R)-naproxen,
(S)-naproxen; (R,S)-ketoprofen, (R)-ketoprofen, (S)-ketoprofen can
be used.
[0009] It is intended by the term "derivative" any compound of the
invention derived by chemical substitutions on its chemical
backbone.
[0010] In a preferred embodiment the anti-inflammatory compound is
a methylxanthine anti-inflammatory compound.
[0011] In another particular embodiment the nitric oxide releasing
compound is isosorbide mononitrate and the anti-inflammatory
compound is aspirin.
[0012] In yet another particular embodiment the nitric oxide
releasing compound is isosorbide dinitrate and the
anti-inflammatory compound is ibuprofen, as R or S enantiomer or as
racemate.
[0013] Preferably the muscular dystrophy is selected from the group
of: Duchenne muscular dystrophy, Becker muscular dystrophy,
facioscapulohumeral muscular dystrophy, myotonic muscular
dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular
dystrophy, Emery-Dreifuss muscular dystrophy, distal muscular
dystrophy or congenital muscular dystrophy.
[0014] It is an object of the present invention a pharmaceutical
composition comprising a therapeutically effective amount of at
least one nitric oxide releasing compound and at least one
anti-inflammatory compound as defined above and suitable diluents
and/or excipients and/or adjuvants and/or emollients. The
pharmaceutical composition is used for the prophylaxis and/or
treatment of muscular dystrophy as defined above. These
pharmaceutical compositions can be formulated in combination with
pharmaceutically acceptable carriers, excipients, stabilizers,
diluents or biologically compatible vehicles suitable for
administration to a subject (for example, physiological saline).
Pharmaceutical composition of the invention include all
compositions wherein said compounds are contained in
therapeutically effective amount, that is, an amount effective to
achieve the medically desirable result in the treated subject. The
pharmaceutical compositions may be formulated in any acceptable way
to meet the needs of the mode of administration. The use of
biomaterials and other polymers for drug delivery, as well the
different techniques and models to validate a specific mode of
administration, are disclosed in literature. Any accepted mode of
administration can be used and determined by those skilled in the
art. For example, administration may be by various parenteral
routes such as subcutaneous, intravenous, intradermal,
intramuscular, intraperitoneal, intranasal, transdermal, oral, or
buccal routes. Parenteral administration can be by bolus injection
or by gradual perfusion over time. Preparations for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions, which may contain auxiliary agents or
excipients known in the art, and can be prepared according to
routine methods. In addition, suspension of the active compounds as
appropriate oily injection suspensions may be administered.
Suitable lipophilic solvents or vehicles include fatty oils, for
example, sesame oil, or synthetic fatty acid esters, for example,
sesame oil, or synthetic fatty acid esters, for example,
ethyloleate or triglycerides. Aqueous injection suspensions that
may contain substances increasing the viscosity of the suspension
include, for example, sodium carboxymethyl cellulose, sorbitol,
and/or dextran. Optionally, the suspension may also contain
stabilizers. Pharmaceutical compositions include suitable solutions
for administration by injection, and contain from about 0.01 to 99
percent, preferably from about 20 to 75 percent of active compound
together with the excipient. Compositions which can be administered
rectally include suppositories. It is understood that the dosage
administered will be dependent upon the age, sex, health, and
weight of the recipient, kind of concurrent treatment, if any,
frequency of treatment, and the nature of the effect desired. The
dosage will be tailored to the individual subject, as is understood
and determinable by one of skill in the art. The total dose
required for each treatment may be administered by multiple doses
or in a single dose. The pharmaceutical composition of the present
invention may be administered alone or in conjunction with other
therapeutics directed to the condition, or directed to other
symptoms of the condition. Usually a daily dosage of active
ingredients is comprised between 0.001 to 100 milligrams per
kilogram of body weight. The compounds of the present invention may
be administered to the patient intravenously in a pharmaceutical
acceptable carrier such as physiological saline. Standard methods
for intracellular delivery of peptides can be used, e.g. delivery
via liposomes. Such methods are well known to those of ordinary
skill in the art. The formulations of this invention are useful for
parenteral administration, such as intravenous, subcutaneous,
intramuscular, and intraperitoneal. As well known in the medical
arts, dosages for any one patient depends upon many factors,
including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently.
[0015] It is a further object of the present invention a diet
supplement comprising an effective amount of at least one nitric
oxide releasing compound and at least one anti-inflammatory
compound as defined above and diluents and/or adjuvants and/or
eccipients.
[0016] Another object of the invention is a method of treating or
preventing muscular dystrophy comprising administering the
pharmaceutical composition or the diet supplement of the
invention.
[0017] The invention will be now described by means of non limiting
examples referring to the following figures:
[0018] FIG. 1: alpha-sarcoglycan (SG)-null C57BL/6 mice were
treated for 30 days with the indicated drugs, or combinations of
drugs incorporated into the diet as described in the Methods. Serum
creatin kinase (CK) concentrations were measured from mouse blood
samples obtained by tail vein withdrawal using the indirect
colorimetric assay (Roche Diagnostics), following standard
procedures. Values shown are the results of 4 experiments
.+-.S.E.M. *P<0.01 vs untreated .alpha.-SG null control mice;
.sctn. P<0.05 vs prednisolone-treated .alpha.-SG null mice.
[0019] FIG. 2: alpha-SG-null C57BL/6 mice were treated for 30 days
with the indicated drugs, or combinations of drugs incorporated
into the diet as described in the Methods. Locomotor performance
was measured through the Running Wheel Activity, to this end mice
were housed individually in cages equipped with a Trixie running
wheel. Each wheel revolution was registered through a magnetic
switch, which was connected to a counter. The number of revolutions
was recorded daily for 6 days. Values shown are the results of 4
experiments .+-.S.E.M. *P<0.01 vs untreated .alpha.-SG null
control mice; .sctn. P<0.05 vs prednisolone-treated .alpha.-SG
null mice. The results of 9 experiments .+-.S.E.M of Running Wheel
Activity measured in wild type (wt) C57BL/6 mice are also
reported.
[0020] FIG. 3: alpha-SG-null C57BL/6 mice were treated for 30 days
with the indicated drugs, or combinations of drugs incorporated
into the diet as described in the Methods. Tibialis anterior
muscles were dissected and frozen in liquid nitrogen-cooled
isopentane. The number of inflammatory infiltrates was measured on
Azan-Mallory-stained serial muscle sections. Values shown are the
results of 4 experiments .+-.S.E.M. *P<0.01 and
.degree.P<0.05 vs untreated .alpha.-SG null control mice.
[0021] FIG. 4: alpha-SG-null C57BL/6 mice were treated for 30 days
with the indicated drugs, or combinations of drugs incorporated
into the diet as described in the Methods. Diaphragm muscles were
dissected, frozen in liquid nitrogen-cooled isopentane and stained
with hematoxilin & eosin according to standard procedures.
Shown are the numbers of necrotic and centronucleated fibers in 4
animals per group .+-.S.E.M. *P<0.01 vs untreated .alpha.-SG
null control mice; .sctn. P<0.05 vs prednisolone-treated
.alpha.-SG null mice.
[0022] FIG. 5: alpha-SG-null C57BL/6 mice were treated for 30 days
with the indicated drugs, or combinations of drugs incorporated
into the diet as described in the Methods. Shown are representative
histological images of diaphragm muscles cross cryosections stained
with in hematoxilin & eosin (A) or Azan Mallory (B) obtained as
described in FIGS. 4 and 3, respectively. Arrows in panels A1-8
point to fiber necrosis, arrows in panels B1-8 point to
inflammatory infiltrates.
[0023] FIG. 6: Satellite cells from alpha-SG-null C57BL/6 mice were
isolated from 3-5 days old mice and induced to differentiate in the
presence of the indicated drugs as described in the methods. Fusion
index was determined at 48 and 72 h as the number of nuclei in
sarcomeric myosin-expressing cells with more than 2 nuclei vs the
total number of nuclei. Reported are the results .+-.S.E.M in 4
experiments. *P<0.01 and .degree.P<0.05 vs untreated
.alpha.-SG null control mice, .sctn. P<0.05 vs
prednisolone-treated .alpha.-SG null mice.
METHODS
Animal Model
[0024] The alpha-SG-null C57BL/6 mouse, one of the most severe
models of muscular dystrophy was used [10,11]. In some experiments
wild type (wt) C57BL/6 mouse were used as internal further
control.
Drugs
[0025] Aspirin (SIGMA, A2093), Ibuprofen, (SIGMA, 17905),
isosorbide-mononitrate (ISMN, Science Lab Com SLI1024), and
isosorbide dinitrate (ISDN, Alexis, 400-800), Prednisolone (SIGMA,
P6254).
[0026] In the in vivo experiments the drugs were administered daily
in the diet to three months old animals for 30 days. The dose of
aspirin was 80 mg/kg, the dose of ISMN was 35 mg/kg and that of
ISDN was 10 mg/kg, while the dose of ibuprofen was 15 mg/kg.
Prednisolone was used as reference drug at the dose of 2 mg/kg.
When combinations of drugs were used, the drugs were both
incorporated in the same diet. Control animals received the same
diet without any drug incorporation.
[0027] In the in vitro experiments, the drugs were applied to
satellite cells at the concentration of 100 .mu.M for ISMN, 50
.mu.M for ISDN, 10 .mu.M for ibuprofen, 50 .mu.M for aspirin and 30
.mu.M for prednisolone. When combinations of drugs were used, the
drugs were administered at the same time.
Creatine Kinase Activity Measurements
[0028] Quantitative determination of creatine kinase activity in
serum of control and drug treated-animals was measured using
creatine kinase reagent (Randox, CK110), according to the
manufacturer's instructions. Blood was collected from tail vein and
serum obtained after centrifugation at 13000 rpm for 10 minutes was
stored at -80.degree. C. before measurements [13].
Free Wheel Running
[0029] Voluntary wheel running was used as the exercise paradigm to
avoid any physiological changes that may occur due to the stress of
forced treadmill running. Mice were housed individually in cages
equipped with a Trixie running wheel. Each wheel revolution was
registered through a magnetic switch, which was connected to a
counter. The number of revolutions was recorded daily for 6 days
[13].
Histology
[0030] Diaphragm and tibialis anterior of untreated and
drug-treated mice were isolated and included in Killik.RTM. frozen
section medium, quickly frozen and cut into 8-.mu.m thick sections
with the muscle fibres oriented transversely using a cryostat.
Sections were stained with either Hematoxylin&Eosin or Azan
Mallory, to evaluate the number of inflammatory infiltrates and
necrotic fibres (18-10 sections per tissue), respectively [10].
Cell Culture and Fusion
[0031] Satellite cells were isolated from 3-5 days old mice as
described previously with some modifications [12]. In particular,
after 3 days from isolation, proliferating myoblasts were
harvested, counted, and plated on tissue culture plastic dishes
coated with 1 mg/ml type I collagen (SIGMA, C9791). After 2 days of
proliferation in growth medium, myogenic cells accounted for more
than 90% of the cultures as revealed by anti-desmin immunostaining
assay. Preparations showing less than 90% myogenic cells were
discarded. Myoblasts were shifted to differentiating medium in the
presence/absence of drugs as indicated in the Results. Growth
medium contained IMDM medium supplemented with 20% Fetal Bovine
Serum, 3% Chick Embryo Extract, 100 U/ml Penicillin, 100 .mu.g/ml
Streptomycin, 50 .mu.g/ml Gentamycin. Differentiation medium
contained IMDM medium supplemented with 2% Horse Serum, 100 U/ml
Penicillin, 100 .mu.g/ml Streptomycin. Fusion index was determined
as the number of nuclei in sarcomeric myosin-expressing cells with
more than 2 nuclei vs the total number of nuclei [10].
Statistical Analyses
[0032] Student's t test for unpaired variables (two tailed) was
used. P<0.05 was considered significant.
Results
In Vivo Experiments
[0033] The effects of the different drugs and their combination on
the number of inflammatory infiltrates, the number of necrotic
fibers per section, creatine kinase activity and free wheel
distance ran are shown in Table I.
TABLE-US-00001 TABLE I Effect of drugs and their combination
Histology data N.sup.o of Free wheel Days of inflammatory N.sup.o
of necrotic CK level test treatment infiltrates/sections
fibres/section (U/l) (km/24 h) Control 30 195.2 .+-. 41.5 272.2
.+-. 37.0 970 .+-. 86 0.13 .+-. 0.02 Aspirin 30 122.2 .+-.
17.1.degree. 249.0 .+-. 32.1 841 .+-. 33 0.15 .+-. 0.02 Ibuprofen
30 116.1 .+-. 11.1.degree. 269.0 .+-. 34.3 851 .+-. 41 0.12 .+-.
0.01 ISMN 30 190.2 .+-. 33.2 238.0 .+-. 27.2 787 .+-. 33 0.18 .+-.
0.03 ISDN 30 180.2 .+-. 18.2 248.0 .+-. 12.1 737 .+-. 45 0.15 .+-.
0.02 Aspirin plus 30 88.2 .+-. 10.5*+ 46.3 .+-. 2.8*+.sctn. 472.7
.+-. 48.9*+.sctn. 0.77 .+-. 0.1*+.sctn. ISMN ISDN plus 30 80.2 .+-.
6.5*+ 38.3 .+-. 2.8*+.sctn. 389.7 .+-. 19.1*+.sctn. 0.84 .+-.
0.2*+.sctn. ibuprofen Prednisolone 30 103.4 .+-. 12.0* 217.3 .+-.
37.3* 757.6 .+-. 37.5 0.38 .+-. 0.1* n = 4 in each group. *P <
0.01; .degree.P < 0.05 vs control; +P < 0.05 vs aspirin or
ibuprofen; .sctn.P < 0.05 vs prednisolone, wt mice: 2.1 .+-. 0.3
Km/24 h in the free wheel run test. CK level values in wt animals
were 82 .+-. 7.5
[0034] The authors found that administration of aspirin, ibuprofen,
ISMN and ISDN alone did not have significant effects on any of the
parameters measured.
[0035] By contrast, the combination of aspirin and ISMN and the
combination of ibuprofen and ISDN, were significantly effective in
reducing the histological, functional and biochemical alterations
observed in these animals (Table I and FIGS. 1-5).
[0036] In particular, serum levels of creatine kinase, a hallmark
of muscle damage, were significantly lower in combination-treated
animals (Table I, FIG. 1) when compared to control animals
receiving the diet without any drug incorporation. Consistently,
animals treated by drug combination performed significantly better
on the free-wheel running test (Table I, FIG. 2) when compared to
control and prednisolone-treated animals. In addition, the combined
treatment aspirin plus ISMN and ibuprofen plus ISDN treatments on
the free-wheel running test improved significantly free wheel
running when compared to the reference compound prednisolone.
[0037] Moreover, animals treated with the drug combinations showed
significantly reduced inflammatory infiltrates (Table I and FIG. 3,
FIG. 5).
[0038] In addition, fibre necrosis was almost undetectable in
animals treated with drug combinations (Table I and FIG. 4, FIG.
5). FIG. 5 reports representative images showing the reduction in
inflammatory infiltrates (panels B1-8) and fibre necrosis (panel
A1-8) induced by the drug combinations as indicated by the
arrows.
In Vitro Experiments
[0039] Satellite cells isolated from newborn alpha-SG-null C57BL/6
mice and plated at low density (6.times.10.sup.3 cells/cm.sup.2)
were maintained for 48 h in growth medium, and then switched to the
differentiating medium in the presence or absence of ISMN, ISDN,
aspirin, ibuprofen, ISMN plus aspirin or ISDN plus ibuprofen. The
fusion index was measured after 48 and 72 h, results are shown
Table II AND FIG. 6.
TABLE-US-00002 TABLE II Effect of drugs and their combination on
fusion index in satellite cells hours of treatment Fusion index (%)
Control 48 21 .+-. 1.3 (n = 4) 72 45 .+-. 2.5 aspirin 48 20 .+-.
1.9 (n = 4) 72 43 .+-. 1.7 Ibuprofen 48 21 .+-. 1.2 (n = 4) 72 42
.+-. 2.3 ISMN 48 41 .+-. 2.2*.sctn. (n = 4) 72 76 .+-. 3.3*.sctn.
ISDN 48 41 .+-. 2.1*.sctn. (n = 4) 72 76 .+-. 3.1*.sctn. Aspirin
plus 48 .sup. 52 .+-. 3.1*.degree..sctn. ISMN 72 .sup. 93 .+-.
4.6*.degree..sctn. (n = 4) Ibuprofen plus 48 .sup. 55 .+-.
2.0*.degree..sctn. ISDN 72 .sup. 102 .+-. 4.9*.degree..sctn. (n =
4) Prednisolone 48 18 .+-. 0.9 (n = 4) 72 41 .+-. 2.0 *P < 0.01
vs control; .degree.P < 0.05 vs ISMN or ISDN, .sctn.P < 0.01
vs prednisolone
[0040] The authors found that the combinations of aspirin and ISMN
and of ibuprofen and ISDN significantly increased the fusion index
when compared to control cells (Table II and FIG. 6). ISMN and ISDN
were effective on their own but less than when combined with
aspirin or ibuprofen (statistical significance values: *P<0.01
vs control; .degree.P<0.05 vs ISMN or ISDN). ISMN and ISDN alone
or combined with aspirin and ibuprofen yielded fusion index values
significantly greater than those obtained with prednisolone
(.sup..sctn.P<0.01 vs prednisolone). To observe an enhanced
satellite fusogenic properties is relevant in the therapeutic
perspective to muscular dystrophies since enhanced fusion of
satellite cells to damaged fibers implies a faster and more
efficient repair of dystrophic damaged fibers.
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